Recent progress and potential future directions to enhance biological nitrogen fixation in faba bean (Vicia faba L.).

The necessity for sustainable agricultural practices has propelled a renewed interest in legumes such as faba bean (Vicia faba L.) as agents to help deliver increased diversity to cropped systems and provide an organic source of nitrogen (N). However, the increased cultivation of faba beans has proven recalcitrant worldwide as a result of low yields. So, it is hoped that increased and more stable yields would improve the commercial success of the crop and so the likelihood of cultivation. Enhancing biological N fixation (BNF) in faba beans holds promise not only to enhance and stabilize yields but also to increase residual N available to subsequent cereal crops grown on the same field. In this review, we cover recent progress in enhancing BNF in faba beans. Specifically, rhizobial inoculation and the optimization of fertilizer input and cropping systems have received the greatest attention in the literature. We also suggest directions for future research on the subject. In the short term, modification of crop management practices such as fertilizer and biochar input may offer the benefits of enhanced BNF. In the long term, natural variation in rhizobial strains and faba bean genotypes can be harnessed. Strategies must be optimized on a local scale to realize the greatest benefits. Future research must measure the most useful parameters and consider the economic cost of strategies alongside the advantages of enhanced BNF.

More than a meat- or synthetic nitrogen fertiliser-substitute: a review of legume phytochemicals as drivers of 'One Health' via their influence on the functional diversity of soil- and gut-microbes.

Legumes are essential to healthy agroecosystems, with a rich phytochemical content that impacts overall human and animal well-being and environmental sustainability. While these phytochemicals can have both positive and negative effects, legumes have traditionally been bred to produce genotypes with lower levels of certain plant phytochemicals, specifically those commonly termed as 'antifeedants' including phenolic compounds, saponins, alkaloids, tannins, and raffinose family oligosaccharides (RFOs). However, when incorporated into a balanced diet, such legume phytochemicals can offer health benefits for both humans and animals. They can positively influence the human gut microbiome by promoting the growth of beneficial bacteria, contributing to gut health, and demonstrating anti-inflammatory and antioxidant properties. Beyond their nutritional value, legume phytochemicals also play a vital role in soil health. The phytochemical containing residues from their shoots and roots usually remain in-field to positively affect soil nutrient status and microbiome diversity, so enhancing soil functions and benefiting performance and yield of following crops. This review explores the role of legume phytochemicals from a 'one health' perspective, examining their on soil- and gut-microbial ecology, bridging the gap between human nutrition and agroecological science.

Nitrogen fixation by common beans in crop mixtures is influenced by growth rate of associated species.

BACKGROUND: Legumes can fix atmospheric nitrogen (N) and facilitate N availability to their companion plants in crop mixtures. However, biological nitrogen fixation (BNF) of legumes in intercrops varies largely with the identity of the legume species. The aim of our study was to understand whether BNF and concentration of plant nutrients by common bean is influenced by the identity of the companion plant species in crop mixtures. In this greenhouse pot study, common beans were cultivated with another legume (chickpea) and a cereal (Sorghum). We compared BNF, crop biomass and nutrient assimilation of all plant species grown in monocultures with plants grown in crop mixtures. RESULTS: We found beans to exhibit low levels of BNF, and to potentially compete with other species for available soil N in crop mixtures. The BNF of chickpeas however, was enhanced when grown in mixtures. Furthermore, biomass, phosphorous and potassium values of chickpea and Sorghum plants were higher in monocultures, compared to in mixtures with beans; suggesting competitive effects of beans on these plants. Concentration of calcium, magnesium and zinc in beans was higher when grown with chickpeas than with Sorghum. CONCLUSIONS: It is generally assumed that legumes benefit their companion plant species. Our study highlights the contrary and shows that the specific benefits of cereal-legume mixtures are dependent on the growth rate of the species concerned. We further highlight that the potential of legume-legume mixtures is currently undervalued and may play a strong role in increasing N use efficiency of intercrop-based systems.

Hydroponic Common-Bean Performance under Reduced N-Supply Level and Rhizobia Application.

This study aims to explore the possibility of a reduced application of inorganic nitrogen (N) fertiliser on the yield, yield qualities, and biological nitrogen fixation (BNF) of the hydroponic common bean (Phaseolus vulgaris L.), without compromising plant performance, by utilizing the inherent ability of this plant to symbiotically fix N2. Until the flowering stage, plants were supplied with a nutrient solution containing N-concentrations of either a, 100%, conventional standard-practice, 13.8 mM; b, 75% of the standard, 10.35 mM; or c, 50% of the standard, 6.9 mM. During the subsequent reproductive stage, inorganic-N treatments b and c were decreased to 25% of the standard, and the standard (100% level) N-application was not altered. The three different inorganic-N supply treatments were combined with two different rhizobia strains, and a control (no-inoculation) treatment, in a two-factorial experiment. The rhizobia strains applied were either the indigenous strain Rhizobium sophoriradicis PVTN21 or the commercially supplied Rhizobium tropici CIAT 899. Results showed that the 50-25% mineral-N application regime led to significant increases in nodulation, BNF, and fresh-pod yield, compared to the other treatment, with a reduced inorganic-N supply. On the other hand, the 75-25% mineral-N regime applied during the vegetative stage restricted nodulation and BNF, thus incurring significant yield losses. Both rhizobia strains stimulated nodulation and BNF. However, the BNF capacity they facilitated was suppressed as the inorganic-N input increased. In addition, strain PVTN21 was superior to CIAT 899-as 50-25% N-treated plants inoculated with the former showed a yield loss of 11%, compared to the 100%-N-treated plants. In conclusion, N-use efficiency optimises BNF, reduces mineral-N-input dependency, and therefore may reduce any consequential negative environmental consequences of mineral-N over-application.

Supply Chain Perspectives on Breeding for Legume-Cereal Intercrops.

Compared to sole crops, intercropping-especially of legumes and cereals-has great potential to improve crop yield and resource use efficiency, and can provide many other ecosystem services. However, the beneficial effects of intercrops are often greatly dependent on the end use as well as the specific species and genotypes being co-cultivated. In addition, intercropping imposes added complexity at different levels of the supply chain. While the need for developing crop genotypes for intercropping has long been recognized, most cultivars on the market are optimized for sole cropping and may not necessarily perform well in intercrops. This paper aims to place breeding targets for intercrop-adapted genotypes in a supply chain perspective. Three case studies of legumes and cereals intercropped for human consumption are used to identify desirable intercrop traits for actors across the supply chains, many of which are not targeted by traditional breeding for sole crops, including certain seed attributes, and some of which do not fit traditional breeding schemes, such as breeding for synchronized maturity and species synergies. Incorporating these traits into intercrop breeding could significantly reduce complexity along the supply chain. It is concluded that the widespread adoption and integration of intercrops will only be successful through the inclusion and collaboration of all supply chain actors, the application of breeding approaches that take into account the complexity of intercrop supply chains, and the implementation of diversification strategies in every process from field to fork.

Global DNA methylation and cellular 5-methylcytosine and H4 acetylated patterns in primary and secondary dormant seeds of Capsella bursa-pastoris (L.) Medik. (shepherd's purse).

Despite the importance of dormancy and dormancy cycling for plants' fitness and life cycle phenology, a comprehensive characterization of the global and cellular epigenetic patterns across space and time in different seed dormancy states is lacking. Using Capsella bursa-pastoris (L.) Medik. (shepherd's purse) seeds with primary and secondary dormancy, we investigated the dynamics of global genomic DNA methylation and explored the spatio-temporal distribution of 5-methylcytosine (5-mC) and histone H4 acetylated (H4Ac) epigenetic marks. Seeds were imbibed at 30 °C in a light regime to maintain primary dormancy, or in darkness to induce secondary dormancy. An ELISA-based method was used to quantify DNA methylation, in relation to total genomic cytosines. Immunolocalization of 5-mC and H4Ac within whole seeds (i.e., including testa) was assessed with reference to embryo anatomy. Global DNA methylation levels were highest in prolonged (14 days) imbibed primary dormant seeds, with more 5-mC marked nuclei present only in specific parts of the seed (e.g., SAM and cotyledons). In secondary dormant seeds, global methylation levels and 5-mC signal where higher at 3 and 7 days than 1 or 14 days. With respect to acetylation, seeds had fewer H4Ac marked nuclei (e.g., SAM) in deeper dormant states, for both types of dormancy. However, the RAM still showed signal after 14 days of imbibition under dormancy-inducing conditions, suggesting a central role for the radicle/RAM in the response to perceived ambient changes and the adjustment of the seed dormancy state. Thus, we show that seed dormancy involves extensive cellular remodeling of DNA methylation and H4 acetylation.

Fresh Pod Yield, Physical and Nutritional Quality Attributes of Common Bean as Influenced by Conventional or Organic Farming Practices.

The aim of the current study wat to comparatively assess the impact of different nitrogen (N) fertilization schemes on fresh pod yield and yield quality in either organically or conventionally grown common beans (Phaseolus vulgaris L.). Prior to common bean crop establishment, the experimental field site was cultivated following either organic (a) or conventional (b) farming practices with a winter non-legume crop (Brassica oleracea var. italica) (BR), or (c) with field bean (Vicia faba sp.) destined to serve as a green manure (GM) crop. At the end of the winter cultivation period the broccoli crop residues (BR) and green manure biomass (GM) were incorporated into the soil and the plots that accommodated the treatments (a) and (c) were followed by an organically cultivated common bean crop, while the conventional broccoli crop was followed by a conventionally cropped common bean crop. Additional to the plant residues (BR), sheep manure (SM) at a rate of 40 kg N ha-1 was also applied to the organically treated common beans, while the plots with a conventionally cropped common bean received 75 kg N ha-1. Organic common bean treated with SM + BR produced smaller pods of higher dry matter and bioactive compound content, responses that are correlated with limited soil N availability. No significant variations were observed on yield components and N levels of pods cultivated under organic (SM + GM) and conventional cropping systems. Pod sugar and starch content was not influenced by the different fertilization practices. In conclusion, we have demonstrated that the combined application of SM + GM can be considered as an efficient N-fertilisation strategy for organic crops of common bean, benefiting their nutritional value without compromising yield.

Differential Interspecific Adaptation to Abiotic Stress by Plantago Species.

The success of seed-based conservation and restoration efforts using native plant species is largely determined by ensuring two key life history transitions are accommodated. These are from "seed to germinated seed" and "germinated seed to established seedling." In turn, optimization of these life history transitions is determined by a "genetic × environmental" interaction and later largely characterized by localized climatic (abiotic) conditions. It is these environmental stress factors that can act as natural selection agents for specific plant-trait combinations, or phenotypes. In turn, such adaptation may also limit a species range. To test the relationship between these two early plant life history stage transitions, "seed to germinated seed" and "germinated seed to established seedling," the attributes were characterized for two species of Plantago that occupy contrasting environments and since these species have potential for native seed-based habit restoration and conservation. The species were Plantago coronopus (L.), localized at lower and drier altitudes, and Plantago lanceolata (L.), characterized as occupying higher and wetter altitudinal clines. Seeds were collected from 20 accessions of six natural populations spanning four European countries for both P. lanceolata and P. coronopus. Seed germination (G) and seedling establishment (S) data were determined at six temperatures (T) and six water potentials (Ψ), and the data obtained were analyzed using a generalized linear model (GLM). The results indicate that P. coronopus has adapted physiologically to its high-altitude conditions such that seed germination and seedling establishment may be more readily achieved in this cooler environment where water is less limiting. In contrast, the lower θT of P. lanceolata better facilitates more efficient seed germination and seedling establishment in drier and warmer clines of lower altitude. In addition to establishing a genotypic (species) underpin for seed and seedling trait differences observed, the insights gained may also be exploited to best deploy each species in situ for seed-based conservation and restoration efforts.

The Push, Pull, and Enabling Capacities Necessary for Legume Grain Inclusion into Sustainable Agri-Food Systems and Healthy Diets.

Legume grains are traditional crops that have been modernized as processed foods and animal protein alternatives in recent years. This modernization has largely been fueled by new technological developments driven by increased consumer demands for plant protein and gluten-free options. However, consumers must be mindful that legumes have other nutritional attributes besides protein that help achieve healthier diets, and recent evidence suggests that consuming 100 g of legume grains per day would promote nutrient-dense diets and could be a target level to harmonize international strategies for recommended daily allowances. The nutritional richness of legumes has been associated with a decrease in numerous disease risk factors and, given their long shelf life, legumes are excellent choices to combat food waste and may have a role to play in countries where cold storage is often a limitation in food preservation. Besides their inclusion in global diets, legumes should be included in sustainable cropping systems. The high number of edible species and cultivars available render them excellent contributors to biodiverse food and feed systems. Legume cultivation allows reducing environmental impacts by means of the ability of legumes to fix atmospheric nitrogen via a symbiosis with nitrogen-fixing bacteria, making them natural fertilizers. Still, despite the well-known health, nutritional, and environmental benefits, legumes are underrepresented in global agri-food systems. Efforts must be made to make legumes more attractive to the farmer, the industry, and ultimately the consumer, and for this proper local, regional, national, and global policy frameworks must be in place. Here, the local scenario of legumes is showcased, and the most relevant push, pull, and enabling capacities required to achieve sustainable diversified agri-food systems with legumes are discussed.

Native Seed Supply and the Restoration Species Pool.

Globally, annual expenditure on ecological restoration of degraded areas for habitat improvement and biodiversity conservation is approximately $18bn. Seed farming of native plant species is crucial to meet restoration goals, but may be stymied by the disconnection of academic research in seed science and the lack of effective policies that regulate native seed production/supply. To illustrate this problem, we identified 1,122 plant species important for European grasslands of conservation concern and found that only 32% have both fundamental seed germination data available and can be purchased as seed. The "restoration species pool," or set of species available in practice, acts as a significant biodiversity selection filter for species use in restoration projects. For improvement, we propose: (1) substantial expansion of research and development on native seed quality, viability, and production; (2) open-source knowledge transfer between sectors; and (3) creation of supportive policy intended to stimulate demand for biodiverse seed.

A Spectrophotometric Assay for Robust Viability Testing of Seed Batches Using 2,3,5-Triphenyl Tetrazolium Chloride: Using Hordeum vulgare L. as a Model.

A comparative analysis was carried out of published methods to assess seed viability using 2,3,5-triphenyltetrazolium chloride (TTC) based assays of seed batches. The tests were carried out on seeds of barley (Hordeum vulgare cv. Optic) as a model. We established that 10% [w/v] trichloroacetic acid (TCA)/methanol is superior to the acetone and methanol-only based methods: allowing the highest recovery of formazan and the lowest background optical density (OD) readings, across seed lots comprising different ratios of viable and dead seeds. The method allowed a linear-model to accurately capture the statistically significant relationship between the quantity of formazan that could be extracted using the method we developed and the seed temperature-response, and seed viability as a function of artificially aged seed lots. Other quality control steps are defined to help ensure the assay is robust and these are reported in a Standard Operating Procedure.

A Comparative Nitrogen Balance and Productivity Analysis of Legume and Non-legume Supported Cropping Systems: The Potential Role of Biological Nitrogen Fixation.

The potential of biological nitrogen fixation (BNF) to provide sufficient N for production has encouraged re-appraisal of cropping systems that deploy legumes. It has been argued that legume-derived N can maintain productivity as an alternative to the application of mineral fertilizer, although few studies have systematically evaluated the effect of optimizing the balance between legumes and non N-fixing crops to optimize production. In addition, the shortage, or even absence in some regions, of measurements of BNF in crops and forages severely limits the ability to design and evaluate new legume-based agroecosystems. To provide an indication of the magnitude of BNF in European agriculture, a soil-surface N-balance approach was applied to historical data from 8 experimental cropping systems that compared legume and non-legume crop types (e.g., grains, forages and intercrops) across pedoclimatic regions of Europe. Mean BNF for different legume types ranged from 32 to 115 kg ha-1 annually. Output in terms of total biomass (grain, forage, etc.) was 30% greater in non-legumes, which used N to produce dry matter more efficiently than legumes, whereas output of N was greater from legumes. When examined over the crop sequence, the contribution of BNF to the N-balance increased to reach a maximum when the legume fraction was around 0.5 (legume crops were present in half the years). BNF was lower when the legume fraction increased to 0.6-0.8, not because of any feature of the legume, but because the cropping systems in this range were dominated by mixtures of legume and non-legume forages to which inorganic N as fertilizer was normally applied. Forage (e.g., grass and clover), as opposed to grain crops in this range maintained high outputs of biomass and N. In conclusion, BNF through grain and forage legumes has the potential to generate major benefit in terms of reducing or dispensing with the need for mineral N without loss of total output.

Rhizobium altiplani sp. nov., isolated from effective nodules on Mimosa pudica growing in untypically alkaline soil in central Brazil.

Root nodule bacteria were isolated from nodules on Mimosa pudica L. growing in neutral-alkaline soils from the Distrito Federal in central Brazil. The 16S rRNA gene sequence analysis of 10 strains placed them into the genus Rhizobium with the closest neighbouring species (each with 99 % similarity) being Rhizobium grahamii, Rhizobium cauense, Rhizobium mesoamericanum and Rhizobium tibeticum. This high similarity, however, was not confirmed by multi-locus sequence analysis (MLSA) using three housekeeping genes (recA, glnII and rpoB), which revealed R. mesoamericanum CCGE 501T to be the closest type strain (92 % sequence similarity or less). Chemotaxonomic data, including fatty acid profiles [with majority being C19 : 0 cyclo ω8c and summed feature 8 (C18 : 1ω7c/C18 : 1ω6c)], DNA G+C content (57.6 mol%), and carbon compound utilization patterns supported the placement of the novel strains in the genus Rhizobium. Results of average nucleotide identity (ANI) differentiated the novel strains from the closest species of the genus Rhizobium, R. mesoamericanum, R. grahamii and R. tibeticum with 89.0, 88.1 and 87.8 % similarity, respectively. The symbiotic genes essential for nodulation (nodC) and nitrogen fixation (nifH) were most similar (99-100 %) to those of R. mesoamericanum, another Mimosa-nodulating species. Based on the current data, these 10 strains represent a novel species of the genus Rhizobium for which the name Rhizobium altiplani sp. nov. is proposed. The type strain is BR 10423T (=HAMBI 3664T).

Improving intercropping: a synthesis of research in agronomy, plant physiology and ecology.

Intercropping is a farming practice involving two or more crop species, or genotypes, growing together and coexisting for a time. On the fringes of modern intensive agriculture, intercropping is important in many subsistence or low-input/resource-limited agricultural systems. By allowing genuine yield gains without increased inputs, or greater stability of yield with decreased inputs, intercropping could be one route to delivering 'sustainable intensification'. We discuss how recent knowledge from agronomy, plant physiology and ecology can be combined with the aim of improving intercropping systems. Recent advances in agronomy and plant physiology include better understanding of the mechanisms of interactions between crop genotypes and species ­ for example, enhanced resource availability through niche complementarity. Ecological advances include better understanding of the context-dependency of interactions, the mechanisms behind disease and pest avoidance, the links between above- and below-ground systems, and the role of microtopographic variation in coexistence. This improved understanding can guide approaches for improving intercropping systems, including breeding crops for intercropping. Although such advances can help to improve intercropping systems, we suggest that other topics also need addressing. These include better assessment of the wider benefits of intercropping in terms of multiple ecosystem services, collaboration with agricultural engineering, and more effective interdisciplinary research.

The rheological properties of the seed coat mucilage of Capsella bursa-pastoris L. Medik. (shepherd's purse).

The outer surface of myxospermous seed coats contains mucilage which absorbs large amounts of water relative to its dry weight. Ecologically, the seed mucilage can affect seed germination and dormancy. Upon hydration, a large proportion of the seed mucilage is lost to the soil and the physics of soil-seed mucilage interactions has not been assessed. Towards that end, the dynamic rheological properties of mucilage extracted from Capsella bursa-pastoris L. Medik. (shepherd's purse) seeds were assessed as a function of mucilage concentration (1-10% [w/w]), temperature (0-80°C) and shear frequency (0.1-100 rad s-1). The seed mucilage was shear thinning and was classified as a highly viscous "weak gel". The relationship between the viscoelastic parameters (viscosity, η*, storage and loss modulus, G' and G″, yield and flow stresses, τy and τf) and mucilage concentration were well fitted by power law models. The values of η*, G' and G″ increased as temperature increased above 40°C and were also slightly frequency dependent. The shepherd's purse seed mucilage is more viscous than that from other plant parts, such as fruits and roots. These properties highlight the possibility that seed mucilage may affect soil conditions and therefore present an additional facilitative ecological role (beyond that already reported, which directly affect seed biology); and this is discussed.

A mathematical model of mucilage expansion in myxospermous seeds of Capsella bursa-pastoris (shepherd's purse).

BACKGROUND AND AIMS: Myxospermy is a term which describes the ability of a seed to produce mucilage upon hydration. The mucilage is mainly comprised of plant cell-wall polysaccharides which are deposited during development of those cells that comprise the seed coat (testa). Myxospermy is more prevalent among those plant species adapted to surviving on arid sandy soils, though its significance in determining the ecological fitness of plants is unclear. In this study, the first mathematical model of myxospermous seed mucilage expansion is presented based on seeds of the model plant species Capsella bursa-pastoris (shepherd's purse). METHODS: The structures underpinning the expansion process were described using light, electron and time-lapse confocal micrographs. The data and experimental observations were used to create a mathematical model of myxospermous seed mucilage expansion based on diffusion equations. KEY RESULTS: The mucilage expansion was rapid, taking 5 s, during which the cell mucilage volume increased 75-fold. At the level of the seed, this represented a 6-fold increase in seed volume and a 2·5-fold increase in seed surface area. These increases were shown to be a function of water uptake (16 g water g(-1) mucilage dry weight), and relaxation of the polymers which comprised the mucilage. In addition, the osmotic pressure of the seed mucilage, estimated by assessing the mucilage expansion of seeds hydrated in solutions of varying osmotic pressure, was -0·54 MPa (equivalent to 0·11 M or 6·6 g L(-1) NaCl). CONCLUSIONS: The results showed that the mucilage may be characterized as hydrogel and seed-mucilage expansion may be modelled using the diffusion equation described. The potential of myxospermous seeds to affect the ecological services provided by soil is discussed briefly.

Co-adaptation of seed dormancy and flowering time in the arable weed Capsella bursa-pastoris (shepherd's purse).

BACKGROUND AND AIMS: The duration of the plant life cycle is an important attribute that determines fitness and coexistence of weeds in arable fields. It depends on the timing of two key life-history traits: time from seed dispersal to germination and time from germination to flowering. These traits are components of the time to reproduction. Dormancy results in reduced and delayed germination, thus increasing time to reproduction. Genotypes in the arable seedbank predominantly have short time to flowering. Synergy between reduced seed dormancy and reduced flowering time would create stronger contrasts between genotypes, offering greater adaptation in-field. Therefore, we studied differences in seed dormancy between in-field flowering time genotypes of shepherd's purse. METHODS: Genotypes with early, intermediate or late flowering time were grown in a glasshouse to provide seed stock for germination tests. Secondary dormancy was assessed by comparing germination before and after dark-incubation. Dormancy was characterized separately for seed myxospermy heteromorphs, observed in each genotype. Seed carbon and nitrogen content and seed mass were determined as indicators of seed filling and resource partitioning associated with dormancy. KEY RESULTS: Although no differences were observed in primary dormancy, secondary dormancy was weaker among the seeds of early-flowering genotypes. On average, myxospermous seeds showed stronger secondary dormancy than non-myxospermous seeds in all genotypes. Seed filling was similar between the genotypes, but nitrogen partitioning was higher in early-flowering genotypes and in non-myxospermous seeds. CONCLUSIONS: In shepherd's purse, early flowering and reduced seed dormancy coincide and appear to be linked. The seed heteromorphism contributes to variation in dormancy. Three functional groups of seed dormancy were identified, varying in dormancy depth and nitrate response. One of these groups (FG-III) was distinct for early-flowering genotypes. The weaker secondary dormancy of early-flowering genotypes confers a selective advantage in arable fields.

Heterogeneity in the distribution of genetically modified and conventional oilseed rape within fields and seed lots.

The implementation of co-existence in the commercialisation of GM crops requires GM and non-GM products to be segregated in production and supply. However, maintaining segregation in oilseed rape will be made difficult by the highly persistent nature of this species. An understanding of its population dynamics is needed to predict persistence and develop potential strategies for control, while to ensure segregation is being achieved, the production of GM oilseed rape must be accompanied by the monitoring of GM levels in crop or seed populations. Heterogeneity in the spatial distribution of oilseed rape has the potential to affect both control and monitoring and, although a universal phenomenon in arable weeds and harvested seed lots, spatial heterogeneity in oilseed rape populations remains to be demonstrated and quantified. Here we investigate the distribution of crop and volunteer populations in a commercial field before and during the cultivation of the first conventional oilseed rape (winter) crop since the cultivation of a GM glufosinate-tolerant oilseed rape crop (spring) three years previously. GM presence was detected by ELISA for the PAT protein in each of three morphologically distinguishable phenotypes: autumn germinating crop-type plants (3% GM), autumn-germinating 'regrowths' (72% GM) and spring germinating 'small-type' plants (17% GM). Statistical models (Poisson log-normal and binomial logit-normal) were used to describe the spatial distribution of these populations at multiple spatial scales in the field and of GM presence in the harvested seed lot. Heterogeneity was a consistent feature in the distribution of GM and conventional oilseed rape. Large trends across the field (50 x 400 m) and seed lot (4 x 1.5 x 1.5 m) were observed in addition to small-scale heterogeneity, less than 20 m in the field and 20 cm in the seed lot. The heterogeneity was greater for the 'regrowth' and 'small' phenotypes, which were likely to be volunteers and included most of the GM plants detected, than for the largely non-GM 'crop' phenotype. The implications of the volunteer heterogeneity for field management and GM-sampling are discussed.

Sources of uncertainty in the quantification of genetically modified oilseed rape contamination in seed lots.

Testing of seed and grain lots is essential in the enforcement of GM labelling legislation and needs reliable procedures for which associated errors have been identified and minimised. In this paper we consider the testing of oilseed rape seed lots obtained from the harvest of a non-GM crop known to be contaminated by volunteer plants from a GM herbicide tolerant variety. The objective was to identify and quantify the error associated with the testing of these lots from the initial sampling to completion of the real-time PCR assay with which the level of GM contamination was quantified. The results showed that, under the controlled conditions of a single laboratory, the error associated with the real-time PCR assay to be negligible in comparison with sampling error, which was exacerbated by heterogeneity in the distribution of GM seeds, most notably at a small scale, i.e. 25 cm3. Sampling error was reduced by one to two thirds on the application of appropriate homogenisation procedures.